Metabolism

• Principal Characteristics of Metabolic Pathways

  • Irreversible
  • Catabolic and Anabolic Pathways Differ
  • Each pathway has a committed step
  • All pathways are regulated
• Hexokinase

  • Found in all body cells
  • Kinase = enzyme that phosphorylates substrate using ATP as the donor
  • Hexo = denotes 6 carbons
  • Glucose-6-phosphate is not substrate for GLUT proteins so is trapped inside cells as it cannot plasma membranes
• Phosphofructokinase

  Catalyses first irreversible step of glycolysis

  Subject to allosteric regulation

  High AMP increases activity

  High ATP decreases activity
• Triose Phosphate Isomerase (TIM)

  • The perfect enzyme
  • Reaction rate is diffusion controlled
  • Product formed as rapidly as substrate and enzyme collide
  • Any increase in catalytic efficiency would not increase reaction rate
  • Two metabolites maintained in equilibrium
  • In isolated enzyme system [DHAP]>>[GAP] but GAP is substrate for next step so more DHAP converted to maintain equilibrium
  • Gibbs is essentially zero - perfect equilibrium
• Why is production of lactate important?

  • Some cells do not have mitochondria so cannot use aerobic oxidation to regenerate NAD
  • Sometimes tissues need to generate energy quicker than aerobic oxidation of NADH can allow
  • Important for maintenance of blood pH
  • Used as primary energy source by some cells
• Committed Step

  • Most component reactions jn a pathway function close to equilibrium - irreversible steps exception
  • Usually an irreversible step early in pathway - shifts the equilibrium of subsequent reactions by increasing substrate concentration
  • Forn ideal point for metabolic regulation - happens early in pathway so metabolites are not diverted into metabolic dead-ends
• Things to consider with Metabolic Pathways

  • The chemical interconversion steps - the sequence of chemical reactions
  • The enzymatic mechanisms
  • Pathway thermodynamics
  • Pathway regulation
• ETC - Functional components

  • Complex 1 - NADH dehydrogenase - more than 20 polypeptides, flavoproteins, iron-sulphur proteins
  • Complex 3 - Cytochrome b/c1 complex - 3-7 polypeptides, cytochrome b, cytochrome c1, Rieke fe/s protein
  • Complex 4 - Cytochrome oxidase - cytochrome a, cytochrome a3, Cu
• Cytochromes

  Similarities

  • Contain a haeme group
  • Undergo 1 electron oxidation/reduction

  Differences

  • Cyt a,b,c distinguished by absorbance spectra
  • A.b haeme is non-covalently bonded to protein (c is covalently bound)
  • Span a range of redox potentials
  • Apoproteins - cyt a,b = integral membrane proteins. Cyt c = extrinsic membrane proteins
• Ubiquinone

  • Only electron carrier that is not bound to protein
  • Oxidation/reduction involves 2 H transfer
  • UQ + 2H+ + 2e- = UQH2 ( ubiquinone to ubiquinol)
• Glycolysis: Step 1

  alpha-D-glucose to glucose-6-phosphate

  Catalysed by hexokinase - glucose binding causes conformational change bringing ATP closer to the hydroxyl group on carbon 6 excluding water

  Irreversible

  ATP to ADP and Pi
• Glycolysis Step 2

  Glucose-6-phosphate to fructose 6-phosphate

  Catalysed by phosphoglucose isomerase

  Readily reversible
• Glycolysis Step 3

  Fructose 6-phosphate to fructose 1,6-bisphosphate

  Catalysed by phosphofructokinase - primary control

  ATP to ADP and Pi

  Irreversible because delta G highly negative

  Committed step

  Highly regulated, allosteric regulation

  High ATP decreases activity, high AMP increases activity
• Allosteric regulation

  Small molecule binds somewhere on the enzyme away from the active site
• Glycolysis Step 4

  Fructose 1,6-bisphosphate to GAP or DAP

  Catalysed by aldolase
• Glycolysis Step 5

  DAP (dihydroxyacetone phosphate) interconverts to GAP (glyceraldehyde-3-phosphate)

  DAP needs to be converted to enediol intermediate

  Both are phosphotrioses

  Catalysed by triose-phosphate isomerase

  In isolated system [DAP] much greater than [GAP] but in glycolysis, [GAP] decreases because it is used up
• 
  

  Glycolysis step 6

  Glyceraldehyde 3 phosphate oxidised to 1,3-bisphosphoglycerate

  Catalysed by GAPDH

  NAD to NADH and H

  Requires free floating phosphate ion
• Glycolysis Step 7

  1,3-bisphosphate glycerate to 3-phosphoglycerate

  Catalysed by phosphoglycerate kinase

  ADP to ATP ( 2 ATP made per glucose)

  Substrate level phosphorylation
• Glycolysis Step 9

  3-phosphoglycerate to 2-phosphoglycerate

  Catalysed by phosphoglyceromutase
• Glycolysis Step 10

  2-phosphoglycerate to phosphoenol-pyruvate

  Catalysed by enolase

  Dehydration reaction
• Glycolysis Step 11

  Phosphoenol-pyruvate to pyruvate (pyruvic acid)

  Catalysed by pyruvate kinase

  ADP to ATP

  Happens twice so these are the two ATP molecules released overall for glycolysis
• Red blood cells have no mitochondria so constantly produce lactate

  Neurons also respire using lactate
• Gluconeogenesis Step 1 - Generation of Phosphoenolpyruvate

  Pyruvate has to enter the mitochondria

  Pyruvate to Oxaloacetate

  Catalysed by pyruvate carboxylase

  ATP + CO2 to ADP + Pi

  Oxaloacetate to Phosphoenolpyruvate

  Catalysed by Phosphoenolpyruvate Carboxykinase (PEP-CK)

  GTP to GDP and removes CO2

  In cytosol. PEP-CK inhibited in conditions favouring glycolysis

  Phosphoenolpyruvate to Pyruvate - enzyme inhibited in conditions favouring gluconeogenesis
• 2 processes used to transport oxaloacetate across mitochondrial membranes

  • Oxaloacetate to Malate - Catalysed by malate dehydrogenase. NADH + H to NAD+
  • Oxaloacetate to Aspartate - Catalysed by aspartate aminotransferase. Amino acid turns to alpha-keto acid
  • Malate and Aspartate can pass through the membrane
  • Multiple potential sites of regulation
• Gluconeogenesis Step 2: Generation of Fructose- 6-Phosphate

  Fructose 1,6-bisphosphate to Fructose 6-phosphate

  Pi lost

  Exergonic reaction so is thermodynamically favoured

  Catalysed by fructose bisphosphatase

  Fructose 6-phosphate to Fructose 1,6-bisphosphate

  Catalysed by phosphofructokinase

  ATP to ADP + Pi
• Gluconeogenesis Step 3: Generation of Glucose from Glucose-6-Phosphate

  Glucose to Glucose 6-phosphate

  Catalysed by hexokinase

  ATP to ADP + Pi

  Glucose 6-phosphate to Glucose

  Catalysed by Glucose 6-phosphatase

  enzyme found in liver and kidneys - bound to ER membrane
• Gluconeogenesis: Raw Materials

  • Glycerol
  • Amino Acids - via the TCA cycle and oxaloacetate. Provides carbon skeleton so any amino acid can be used
  • Lactate - via the Cori Cycle. From anaerobically respiring tissues and erythrocytes
• Anaerobic - Lactate

  Pyruvate to Lactate

  NADH + H+ to NAD+

  Reverse is catalysed by lactate dehydrogenase
• Glycerol - from the breakdown of triglycerides

  Glycerol to Glycerol 3-phosphate

  Catalysed by Glycerol kinase. ATP to ADP + Pi

  Glycerol 3-phosphate to Dihydroxyacetone 3-phosphate

  (middle hydroxyl group oxidised to ketone)

  Catalysed by glycerol-3-phosphate dehydrogenase

  NAD+ to NADH
• Link Reaction

  Pyruvate + reduced CoA + NAD+ to Acetyl CoA + CO2 + NADH + H+

  Aerobic respiration only

  Irreversible

  Catalysed by pyruvate dehydrogenase

  Happens in the mitochondria
• Pyruvate Dehydrogenase Complex

  Multienzyme complex

  • Big - more than 10,000 kD
  • one of the largest protein-complexes known

  Complex

  • 3 enzyme activities - 3 enzymatic units, E1, E2, E3
  • 60 polypeptide chains

  Efficient

  • places substrates and active sites in close proximity
  • allows channelling - minimises side reactions
  • allows co-ordinate control
• TCA Cycle - Step 1

  Oxaloacetate + Acetyl CoA to S-Citryl CoA

  Condensation reaction

  S-Citryl CoA to Citrate

  Hydrolysis reaction

  Catalysed by citrate synthase

  Highly negative Gibbs prime so irreversible
• TCA Cycle - Step 2

  Citrate to cis-Aconitate

  Dehydration reaction catalysed by aconitase

  cis-Aconitase to Isocitrate

  Hydration reaction catalysed by aconitase

  Reorganisation. Gibbs practically zero
• TCA Cycle - Step 3

  Isocitrate to Oxalosuccinate

  Oxidation reaction (hydroxyl group on alpha carbon to a ketone)

  Catalysed by isocitrate dehydrogenase

  Oxalosuccinate to alpha-ketoglutarate

  Decarboxylation reaction

  Catalysed by isocitrate dehydrogenase
• TCA Cycle - Step 4

  alpha-ketoglutarate to Succinyl CoA

  COOH replaced with CoA

  Dehydration followed by a decarboxylation

  NAD+ to NADH + H+

  CoASH in, CO2 out

  Catalysed by alpha-ketogluturate dehydrogenase complex (large multi-enzyme complex)

  Irreversible
• TCA Cycle - Step 5

  Succinyl CoA to Succinate

  Breaking high energy bond to use energy to make GTP from GDP and Pi

  Catalysed by Succinyl CoA synthetase
• TCA Cycle - Step 6

  Succinate to Fumurate

  Rearranging step

  Oxidation reaction - FAD is reduced to FADH2

  Catalysed by succinate dehydrogenase (associated with inner mitochondrial membrane)
• TCA Cycle - Step 7

  Fumurate to L-Malate

  Fumurate is a symmetrical molecule

  Catalysed by Fumurase

  Hydration reaction

  Stereospecific addition of water
• TCA Cycle - Step 8

  L-Malate to Oxaloacetate

  Catalysed by Malate dehydrogenase

  NAD+ to NADH + H+

  Unfavourable oxidation driven by citrate synthase reaction
• Anaplerosis = filling up

  Fill up TCA intermediates - important for regulation

  TCA is anabolic because it breaks down molecules to capture energy to make GTP